WO2024012464A1

WO2024012464A1 - Use of imidazotetrazine compound

Info

Publication number: WO2024012464A1
Application number: PCT/CN2023/106847
Authority: WO
Inventors: 尚宝虎; 马静静; 侯晓磊
Original assignee: 北京紫萌医药科技有限公司
Priority date: 2022-07-11
Filing date: 2023-07-11
Publication date: 2024-01-18

Abstract

The present invention relates to new use of an imidazotetrazine compound, or a solvate thereof, a hydrate thereof, a polymorph thereof, a prodrug thereof or a pharmaceutically acceptable salt thereof, and particularly to use in the preparation of a medicament for treating and/or preventing diffuse large B cell lymphoma in a subject.

Description

Uses of imidazotetrazine compounds

Technical field

The present invention belongs to the field of medicine, and more specifically, the present invention relates to new anti-tumor uses of imidazotetrazine compounds, and the use of pharmaceutical preparations at least including them as active ingredients in the prevention and treatment of diffuse lymphoma.

Background technique

According to the "World Health Organization Pathological Classification Criteria for Lymphatic System Tumors", there are currently nearly 70 known pathological types of lymphoma, which can be roughly divided into two categories: Hodgkin's lymphoma and non-Hodgkin's lymphoma. Among them, Hodgkin's lymphoma Lymphoma accounts for about 10% of lymphomas, and non-Hodgkin lymphoma accounts for about 90% of all lymphoma cases, and the incidence rate is increasing year by year. Non-Hodgkin's lymphoma is divided into two categories: B cell type and T/NK cell type, of which B cell type lymphoma accounts for about 70% and T/NK cell type lymphoma accounts for about 30%. With the basic and clinical research With the continuous development, the classification of lymphoma is still further refined and improved.

In addition, diffuse large B lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL), accounting for about 30-40% of all adult NHLs. It can occur in all ages, and is more common in the elderly. Median age 60: slightly more men than women. DLBCL accounts for the majority of cases of clinically "aggressive" or "moderately high malignant" lymphoma. About 40% of DLBCL patients have an average life span of less than 5 years after diagnosis due to relapse or refractory treatment. Therefore, the specificity of DLBCL Research on treatment is of great significance.

According to relevant research reports, DLBCL may be related to congenital inheritance, out-of-control apoptosis mechanism, and DNA repair defects. However, the specific cause of DLBCL is still unclear. Over the past two decades, the clinical efficacy of DLBCL treatment has improved significantly, mainly due to the traditional combination of rituximab and chemotherapy as initial treatment and the refinement of high-dose chemotherapy and autologous stem cell transplantation strategies to treat DLBCL. However, both treatment options have the problem of drug resistance in DLBCL treatment, which affects the clinical application of related drugs. Therefore, new treatment strategies are urgently needed.

The imidazotetrazine compound 3-methyl-4-oxo-8-imidazo[5,1-d][1,2,3,5]tetrazinecarboxamide (TMZ, see U.S. Patent No. 5260291) is A chemotherapeutic agent approved for the treatment of brain tumors (marketed by Schering under the trademark Temodar in the United States and in Europe under the trademark Temodal). Under biological conditions, TMZ degrades via pH-dependent hydrolysis to its active metabolite MTIC (3-methyl-(triazen-1-yl)imidazole-4-carboxamide). Temodar capsules are currently indicated in the United States for the treatment of adult patients with newly diagnosed glioblastoma multiforme and refractory anaplastic astrocytoma, and Temodar capsules are currently approved in Europe for the treatment of patients with malignant glial tumors. tumor. In addition, TMZ has been reported to be used in the treatment of a variety of lymphomas, such as the treatment of T/NK cell type (Ma Xuejun, Fudan Study on the Treatment Patterns, Radiotherapy Plans and Prognostic Related Factors of Early Extranodal NK/T Cell Lymphoma Nasal Type University doctoral thesis), treatment of primary central nervous system lymphoma (O Asif and M Nehal, Temozolomide for Relapsed Primary CNS Lymphoma Journal of the College of Physicians and Surgeons Pakistan 2012,Vol.22(9):594-595 ), treatment of leukemia and lymphoma (Wang Chunmei, study on the synthesis and biological activity of temozolomide derivatives, Guizhou University master's thesis).

Contents of the invention

The object of the present invention is to provide the application of imidazotetrazine compounds, especially TMZ, in the preparation of medicines for preventing or treating diffuse lymphoma. The present invention surprisingly found that imidazotetrazine compounds selectively treat tumors other than the primary site. Or diffuse lymphoma whose metastasis site is the central nervous system has excellent antagonistic effects.

The first aspect of the present invention provides imidazotetrazine compounds, or their solvates, hydrates, polymorphs, prodrugs or pharmaceutically acceptable salts for the preparation of medicaments for the treatment and/or prevention of diffuse lymphoma. Use in the invention, wherein the imidazotetrazine compound is represented by formula I:

In formula I, R ₁ is hydrogen or C1-C3 alkyl, R ₂ is amino or amino substituted by C1-C3 alkyl; R ₃ is hydrogen or C1-C3 alkyl.

In some embodiments, the DLBCL is selected from diffuse large B-cell lymphoma whose primary site and/or metastatic site is not the central nervous system (except for DLBCL whose primary site and/or metastatic site is the central nervous system). ).

In some embodiments, the DLBCL is selected from the group consisting of unspecified DLBCL, other large B-cell lymphoma, high-grade B-cell lymphoma, and unclassifiable B-cell lymphoma.

In some embodiments, the DLBCL is selected from the group consisting of centroblast DLBCL, immunoblastic variant DLBCL, anaplastic DLBCL, spindle cell variant DLBCL, signet ring cell-like variant DLBCL, T cell/histiocyte enriched DLBCL, primary cutaneous DLBCL, EBV-positive DLBCL, chronic inflammation-associated large B-cell lymphoma, lymphomatoid granuloma, large B-cell lymphoma with IRF4 rearrangement, large B-cell lymphoma with IRF4/MUM1 rearrangement cellular lymphoma, primary mediastinal large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, HHV8-positive DLBCL, primary effusion lymphoma, high Grade B-cell lymphoma with MYC, BCL2 and/or BCL6 rearrangements, high-grade B-cell lymphoma not otherwise specified, unclassifiable B-cell lymphoma between DLBCL and classical Hodgkin lymphoma, between primary B-cell lymphoma between mediastinal large B-cell lymphoma and nodular sclerosing classical Hodgkin lymphoma, B-cell lymphoma between DLBCL and Burkitt lymphoma.

In some embodiments, the DLBCL is primary DLBCL, or transformed DLBCL, or advanced and/or relapsed, and/or refractory DLBCL, or DLBCL that has previously received systemic therapy, or DLBCL that has failed chemotherapy drugs and/or targeted drugs and/or immunotherapy.

In some embodiments, the cells of the DLBCL are selected from the group consisting of human diffuse histological lymphoma cell SU-DHL-2, human diffuse lymphoma cell line TMD-8, and human diffuse large cell lymphoma cell SU-DHL-6 one or more of them.

A second aspect of the present invention provides the use of a pharmaceutical composition in the preparation of medicaments for the treatment and/or prevention of DLBCL, the pharmaceutical composition comprising an imidazotetrazine compound represented by Formula I or a solvate thereof , hydrates, polymorphs, prodrugs or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable excipients,

In some embodiments, _R1 is methyl.

In some embodiments, _R2 is amino.

In some embodiments, _R3 is hydrogen.

In some embodiments, R ₁ is methyl, R ₂ is amino, and R ₃ is hydrogen.

In some embodiments, the DLBCL is selected from diffuse large B-cell lymphoma whose primary site and/or metastatic site is not the central nervous system.

A third aspect of the present invention also provides a pharmaceutical combination, including an imidazotetrazine compound and a second therapeutic agent that can be used to treat DLBCL. The imidazotetrazine compound is represented by Formula I:

Among them, R ₁ is hydrogen or C1-C3 alkyl, R ₂ is amino or C1-C3 alkyl substituted amino; R ₃ is hydrogen or C1-C3 alkyl.

In some embodiments, _R1 is methyl.

In some embodiments, _R2 is amino.

In some embodiments, _R3 is hydrogen.

In some embodiments, R ₁ is methyl, R ₂ is amino; R ₃ is hydrogen, i.e., the imidazotetrazine is temozolomide (3-methyl-4-oxo-8-imidazo[5,1 -d][1,2,3,5]tetrazinecarboxamide).

In some embodiments, the second therapeutic agent is one or more of chemotherapy drugs, small molecule targeted anti-tumor drugs, immunotherapy drugs, and antibody drugs. In some preferred embodiments, the second therapeutic agent is cyclophosphamide.

The fourth aspect of the present invention relates to the use of the pharmaceutical combination described in the third aspect in the preparation of medicaments for the treatment and/or prevention of DLBCL.

In some embodiments, the DLBCL is selected from DLBCL other than DLBCL whose primary site and/or metastatic site is the central nervous system.

Another aspect of the present invention relates to a method for treating and/or preventing DLBCL in a subject, the method comprising administering to the subject an effective amount of an imidazotetrazine compound or a solvate, hydrate thereof Materials, polymorphs, prodrugs or pharmaceutically acceptable salts or pharmaceutical combinations thereof, wherein the imidazotetrazine compound is represented by formula I:

Wherein, R ₁ is hydrogen or C1-C3 alkyl, R ₂ is amino or amino substituted by C1-C3 alkyl; R ₃ is hydrogen or C1-C3 alkyl.

In some embodiments, _R1 is methyl.

In some embodiments, _R2 is amino.

In some embodiments, _R3 is hydrogen.

In some embodiments, R ₁ is methyl, R ₂ is amino; R ₃ is hydrogen.

In some embodiments, the imidazotetrazine is 3-methyl-4-oxo-8-imidazo[5,1-d][1,2,3,5]tetrazinecarboxamide.

In some embodiments, the pharmaceutical combination further includes a second therapeutic agent capable of treating DLBCL.

In some embodiments, the second therapeutic agent is one or more of chemotherapy drugs, small molecule targeted anti-tumor drugs, immunotherapy drugs, and antibody drugs. Preferably, the second therapeutic agent is cyclophosphamide.

In some embodiments, the subject is a mammal, preferably a human.

In some embodiments, the DLBCL can be selected from the group consisting of centroblast DLBCL, immunoblastic variant DLBCL, anaplastic DLBCL, spindle cell variant DLBCL, signet ring cell-like variant DLBCL, T cell/histiocyte enrichment DLBCL, primary cutaneous DLBCL, EBV-positive DLBCL, chronic inflammation-associated large B-cell lymphoma, lymphomatoid granuloma, large B-cell lymphoma with IRF4 rearrangement, large B-cell lymphoma with IRF4/MUM1 rearrangement B-cell lymphoma, primary mediastinal large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, HHV8-positive DLBCL, primary effusion lymphoma, High-grade B-cell lymphoma with MYC, BCL2 and/or BCL6 rearrangements, high-grade B-cell lymphoma not otherwise specified, unclassifiable B-cell lymphoma between DLBCL and classical Hodgkin lymphoma, between primary B-cell lymphoma occurs between mediastinal large B-cell lymphoma and nodular sclerosing classical Hodgkin lymphoma, and between DLBCL and Burkitt lymphoma.

In some embodiments, the DLBCL can be primary DLBCL, or transformed DLBCL, or late-stage and/or relapsed, and/or refractory DLBCL, or DLBCL that has previously received systemic therapy, Or DLBCL that has failed chemotherapy drugs and/or targeted drugs and/or immunotherapy. In some embodiments, the cells of the DLBCL are selected from the group consisting of human diffuse histological lymphoma cell SU-DHL-2, human diffuse lymphoma cell line TMD-8, and human diffuse large cell lymphoma cell SU-DHL-6 one or more of them.

Detailed ways

definition

"Subjects" for administration include, but are not limited to: humans (i.e., males or females of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young Adults, middle-aged adults or older adults)) and/or non-human animals, for example, mammals, such as primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, Goats, rodents, cats and/or dogs. In some embodiments, the subject is human. In some embodiments, the subject is a non-human animal. The terms "person," "patient," and "subject" are used interchangeably herein.

"Disease," "disorder," and "condition" are used interchangeably herein.

As used herein, unless otherwise specified, the term "treatment," "treatment," or "treatment" includes an action in a subject suffering from a particular disease, disorder, or condition that reduces the severity of the disease, disorder, or condition, or delays or slows down the disease, disorder, or condition. the development of a disorder or condition ("therapeutic treatment"), and also includes effects that occur before a subject begins to suffer from a specific disease, disorder or condition ("preventive treatment").

Generally, an "effective amount" of a compound is an amount sufficient to elicit a target biological response. As will be understood by those of ordinary skill in the art, the effective amount of a compound of the present invention may vary depending on factors such as, for example, the biological target, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the condition of the subject. Age health conditions and symptoms. The effective amount includes a therapeutically effective amount and a preventive effective amount.

Unless otherwise specified, a "therapeutically effective amount" of a compound as used herein is an amount sufficient to provide a therapeutic benefit in treating a disease, disorder, or condition, or to cause one or more symptoms associated with the disease, disorder, or condition The amount to delay or minimize. A therapeutically effective amount of a compound is that amount of therapeutic agent that, when used alone or in combination with other therapies, provides a therapeutic benefit in the treatment of a disease, disorder, or condition. The term "therapeutically effective amount" may include an amount that improves overall treatment, reduces or avoids symptoms or causes of a disease or disorder, or enhances the therapeutic effect of other therapeutic agents.

Unless otherwise stated, a "prophylactically effective amount" of a compound as used herein is an amount sufficient to prevent a disease, disorder or condition, or to prevent one or more symptoms associated with a disease, disorder or condition, or to prevent a disease , the amount of recurrence of a disorder or condition. A prophylactically effective amount of a compound is that amount of therapeutic agent that, when used alone or in combination with other agents, provides a prophylactic benefit in preventing a disease, disorder, or condition. The term "prophylactically effective amount" may include an amount that improves overall prophylaxis, or an amount that enhances the prophylactic effect of other prophylactic agents.

Those skilled in the art will understand that organic compounds can form complexes with solvents, react in the solvent, or precipitate or crystallize out of the solvent. These complexes are called "solvates". When the solvent is water, the complex is called a "hydrate." This invention encompasses all solvates of the compounds of the invention.

The term "solvate" refers to a form of a compound or a salt thereof that is combined with a solvent, usually formed by a solvolysis reaction. Physical association may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, ether, etc. The compounds described herein can be prepared, for example, in crystalline form, and can be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include stoichiometric and non-stoichiometric solvates. In some cases, the solvate will be capable of isolating, for example, when one or more solvents separate When incorporated into the crystal lattice of a crystalline solid. "Solvate" includes both solution solvates and isolable solvates. Representative solvates include hydrates, ethanolates, and methoxides.

The term "hydrate" refers to a compound combined with water. Typically, the ratio of the number of water molecules contained in a hydrate of a compound to the number of molecules of the compound in the hydrate is determined. Thus, a hydrate of a compound may be represented, for example, by the general formula _R.xH2O , where R is the compound and x is a number greater than zero. A given compound may form more than one hydrate type, including, for example, monohydrate (x is 1), lower hydrate (x is a number greater than 0 and less than 1), for example, hemihydrate (R·0.5H ₂ O)) and polyhydrates (x is a number greater than 1, for example, dihydrate (R·2H ₂ O) and hexahydrate (R·6H ₂ O)).

The compounds of the invention may be in amorphous or crystalline forms (polymorphs). Furthermore, the compounds of the present invention may exist in one or more crystalline forms. Accordingly, the present invention includes within its scope all amorphous or crystalline forms of the compounds of the invention. The term "polymorph" refers to a crystalline form of a compound (or a salt, hydrate or solvate thereof) in a specific crystal packing arrangement. All polymorphs have the same elemental composition. Different crystalline forms often have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optoelectronic properties, stability and solubility. Recrystallization solvent, crystallization rate, storage temperature, and other factors can lead to the dominance of one crystalline form. Various polymorphs of the compounds can be prepared by crystallization under different conditions.

Furthermore, prodrugs are also included within the context of the present invention. The term "prodrug" as used herein refers to a compound that is converted in the body to its active form having a medical effect, for example, by hydrolysis in the blood. Pharmaceutically acceptable prodrugs are described in T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 of A.C.S. Symposium Series, Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, and D. Fleisher, S. Ramon and H. Barbra "Improved oral drug delivery: solubility limitations overcome by the use of prodrugs", Advanced Drug Delivery Reviews (1996) 19 (2) 115-130, which are incorporated into this article as a reference .

A prodrug is any covalently bonded compound of the invention that releases the parent compound in the body when administered to a patient. Prodrugs are typically prepared by modifying functional groups in a manner such that the modification can be cleaved by conventional manipulations or in vivo to yield the parent compound. Prodrugs include, for example, compounds of the invention in which a hydroxyl, amino or thiol group is bonded to any group that can be cleaved to form a hydroxyl, amino or thiol group when administered to a patient. Thus, representative examples of prodrugs include, but are not limited to, amide derivatives of amino functionality that may be present in the compound. In addition, in the case of carboxylic acid (-COOH), esters such as methyl ester, ethyl ester, etc. can be used. The ester itself may be reactive and/or hydrolyzable under human body conditions. Suitable pharmaceutically acceptable in vivo hydrolyzable ester groups include those that readily break down in the human body to release the parent acid or salt thereof.

"Pharmaceutically acceptable" means a pharmaceutical composition that is generally safe, non-toxic and neither biologically or otherwise undesirable for use in the preparation of a pharmaceutical composition, and includes that is acceptable for human pharmaceutical use. accepted. "Pharmaceutically acceptable salts" include, but are not limited to, acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or with organic acids such as acetic acid, trifluoroacetic acid, propionic acid, Caproic acid, heptanoic acid, cyclopentane propionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid Acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, p-chlorobenzenesulfonic acid, p-toluenesulfonic acid, 3-phenylpropionic acid, trimethyl Acid addition salts formed from acetic acid, tert-butylacetic acid, dodecyl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, etc.

Imidazotetrazine compounds

The imidazotetrazine compound described in the present invention is represented by Formula I:

In some embodiments, _R1 is methyl.

In some embodiments, _R2 is amino.

In some embodiments, _R3 is hydrogen.

In some embodiments, R ₁ is methyl, R ₂ is amino; R ₃ is hydrogen, that is, the imidazotetrazine compound is temozolomide.

Pharmaceutical compositions and kits

In another aspect, the present invention provides a pharmaceutical composition comprising the imidazotetrazine compound represented by Formula I of the present invention (also referred to as "active component") and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition contains an effective amount of the imidazotetrazine compound represented by Formula I of the present invention. In some embodiments, the pharmaceutical composition contains a therapeutically effective amount of the imidazotetrazine compound represented by Formula I of the present invention. In some embodiments, the pharmaceutical composition contains a prophylactically effective amount of the imidazotetrazine compound represented by Formula I of the present invention.

Pharmaceutically acceptable excipients for use in the present invention refer to non-toxic carriers, adjuvants or vehicles that do not destroy the pharmacological activity of the compounds with which they are formulated. Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions of the present invention include, but are not limited to, ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins (such as human serum albumin) protein), buffer substances (such as phosphate), glycine, sorbic acid, potassium sorbate, partial glyceride mixture of saturated vegetable fatty acids, water, salt or electrolyte (such as protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate , sodium chloride, zinc salt, silica gel, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, wax, polyethylene-polyoxypropylene- Block polymers, polyethylene glycols, and lanolin.

The present invention also includes kits (eg, pharmaceutical packaging). Kits provided may include a compound of the invention, other therapeutic agents, and first and second containers (e.g., vials, ampoules, bottles, syringes, and/or dispersible packaging or other) containing the compounds of the invention, other therapeutic agents. suitable container). In some embodiments, provided kits may also optionally include a third container containing pharmaceutical excipients for diluting or suspending the compounds of the invention and/or other therapeutic agents. In some embodiments, the compound of the invention and the other therapeutic agent provided in the first container and the second container are combined to form a unit dosage form.

The present invention also provides a pharmaceutical combination, including the above-mentioned imidazotetrazine compound and a second therapeutic agent that can be used to treat DLBCL. In such combinations, the imidazotetrazine compound and the second therapeutic agent may be administered to the subject separately or sequentially.

The second therapeutic agent may be one or more of chemotherapy drugs, small molecule targeted anti-tumor drugs, immunotherapy drugs, and antibody drugs.

Give medication

The compounds or pharmaceutical compositions provided by the invention can be administered through many routes, including but not limited to: oral administration, parenteral administration, inhalation administration, topical administration, rectal administration, nasal administration, oral administration, Vaginal administration, via implant, or other means of administration. example For example, parenteral administration as used herein includes subcutaneous administration, intradermal administration, intravenous administration, intramuscular administration, intraarticular administration, intraarterial administration, intrasynovial administration, and intrasternal administration. , intracerebrospinal membrane drug administration, intralesional drug administration, and intracranial injection or infusion techniques.

Typically, an effective amount of a compound provided herein is administered. The amount of compound actually administered can be determined by the physician depending on the circumstances, including the condition being treated, the route of administration chosen, the compound actually administered, the age, weight and response of the individual patient, the severity of the patient's symptoms, etc. .

When used to prevent a condition described herein, a compound provided herein is administered to a subject at risk of developing the condition, typically on the advice of and under the supervision of a physician, at dosage levels as described above. Subjects at risk of developing a particular condition generally include subjects with a family history of the condition or those who have been determined by genetic testing or screening to be particularly susceptible to developing the condition.

The pharmaceutical compositions provided herein can also be administered over a long period of time ("chronic administration"). Long-term administration refers to the administration of a compound or pharmaceutical composition thereof over a long period of time, for example, 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, etc., or administration may be continued indefinitely, For example, the remainder of the subject's life. In some embodiments, chronic administration is intended to provide a constant level of the compound in the blood over an extended period of time, eg, within a therapeutic window.

Various methods of administration may be used to further deliver the pharmaceutical compositions of the present invention. For example, in some embodiments, a pharmaceutical composition may be administered as a bolus injection, eg, in order to increase the concentration of the compound in the blood to an effective level. The bolus dose depends on the target systemic levels of the active ingredient through the body, e.g., an intramuscular or subcutaneous bolus dose provides a slow release of the active ingredient, whereas a bolus dose delivered directly into the vein (e.g., via an IV drip) ) can be delivered more quickly, allowing the concentration of active ingredients in the blood to quickly increase to effective levels. In other embodiments, the pharmaceutical composition may be administered as a continuous infusion, for example, by IV infusion, thereby providing a steady-state concentration of the active ingredient in the subject's body. Additionally, in other embodiments, a bolus dose of the pharmaceutical composition may be administered first, followed by a continuous infusion.

Oral compositions may take the form of bulk liquid solutions or suspensions, or bulk powders. More typically, however, the compositions are provided in unit dosage form to facilitate precise dosing. The term "dosage unit form" refers to physically discrete units suitable as unitary dosages for human patients and other mammals, each unit containing a predetermined quantity of active material suitable to produce the desired therapeutic effect in association with a suitable pharmaceutical excipient. Typical unit dosage forms include prefilled, premeasured ampoules or syringes for liquid compositions, or pills, tablets, capsules, and the like in the case of solid compositions. In such compositions, the compound will generally be a minor component (from about 0.1 to about 50% by weight, or preferably from about 1 to about 40% by weight), with the remainder being various components useful in forming the desired administration form. carriers or excipients and processing aids.

Liquid forms suitable for oral administration may include suitable aqueous or non-aqueous carriers as well as buffering agents, suspending and dispersing agents, coloring agents, flavoring agents, and the like. Solid forms may include, for example, any of the following components, or compounds of similar nature: binders, such as microcrystalline cellulose, tragacanth, or gelatin; excipients, such as starch or lactose; disintegrants, For example, alginic acid, sodium carboxymethyl starch or corn starch; lubricant, for example, magnesium stearate; glidant, for example, colloidal silicon dioxide; sweetener, for example, sucrose or saccharin; or flavoring agent, for example, Mint, methyl salicylate or orange flavoring; stabilizers such as tartaric acid, aspartic acid, glutamic acid, etc.

Injectable compositions are typically based on injectable sterile saline or phosphate buffered saline, or other injectable excipients known in the art. As stated previously, in such compositions the active compound is typically a minor component, often about 0.05 to 10% by weight, with the remainder being injectable excipients and the like.

Transdermal compositions are typically formulated as topical ointments or creams containing the active ingredients. When formulated as an ointment, the active ingredients are typically combined with a paraffin or water-miscible ointment base. Alternatively, the active ingredient may be formulated as a cream with, for example, an oil-in-water cream base. Such transdermal formulations are well known in the art and often include other components for promoting stable skin penetration of the active ingredient or formulation. All such known transdermal formulations and components are included within the scope provided by this invention.

The compounds of the present invention may also be administered via transdermal devices. Thus, transdermal administration may be achieved using reservoir or porous membrane types, or a variety of solid matrix patches.

The above components of compositions for oral administration, injection or topical administration are merely representative. Other materials and processing techniques are described in Part 8 of Remington's Pharmaceutical Sciences, 17th edition, 1985, Mack Publishing Company, Easton, Pennsylvania, which article is incorporated by reference.

The compounds of the present invention may also be administered in sustained release form or from a sustained release drug delivery system. Descriptions of representative sustained release materials can be found in Remington's Pharmaceutical Sciences.

treat

The imidazotetrazine compounds represented by formula I have value as anti-tumor agents. In particular, the compounds of the invention have value as anti-proliferative, apoptotic and/or anti-invasive agents in the containment and/or treatment of solid and/or liquid tumor diseases. In particular, the compounds of the present invention are expected to be useful in preventing or treating diffuse lymphoma and the like. Anti-cancer effects useful for treating cancer in patients include, but are not limited to, anti-tumor effects, response rates, time to disease progression, and survival rates. The anti-tumor effects of the treatment method of the present invention include, but are not limited to, inhibition of tumor growth, delay of tumor growth, tumor regression, tumor shrinkage, prolonged tumor regeneration after treatment is stopped, and slowing of disease progression. Anticancer effects include preventive treatment as well as treatment of existing disease.

The effective amount of the compound of the present invention is usually at an average daily dose of 0.01 mg to 100 mg compound/kg patient body weight, preferably 0.1 mg to 30 mg compound/kg patient body weight, in single or multiple administrations. Generally, the compounds of the present invention may be administered to the patient in need of such treatment at a daily dosage ranging from about 1 mg to about 3500 mg per patient, preferably 10 mg to 1000 mg. For example, the daily dose per patient may be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 160, 180, 200, 240, 250, 300, 350, 360, 400, 500, 600, 700, 800, 900 or 1000mg. Administration can be one or more times daily, weekly (or several days apart), or on an intermittent schedule. For example, the compound may be administered on a weekly basis (eg, every Monday) one or more times per day, occasionally or for several weeks, eg, 4-10 weeks. Alternatively, the compound may be administered daily for several days (e.g., 2-10 days), followed by several days (e.g., 1-30 days) without administration of the compound, and the cycle may be repeated indefinitely or a given number of times, e.g., 4-10 cycle. For example, a compound of the invention may be administered daily for 5 days, then 9 days off, then again daily for 5 days, then 9 days off, and so on, repeating the cycle indefinitely or a total of 4-10 times.

The compounds of the present invention can be prepared into any dosage form and administered to cancer patients in an appropriate administration method. Those skilled in the art can appropriately select the dosage of the product of the present invention according to the patient's symptoms and physical health. The daily dose can be given as a single dose or divided into multiple doses. Dosage varies based on symptoms, age, etc.

The preferred dosage is, in the case of oral administration to adults, a dosage of 0.1 mg/kg (preferably 0.1 mg/kg) as the lower limit and a dosage of 20 mg/kg (preferably 10 mg/kg) as the upper limit. In the case of enteral administration, the dose is 0.02 mg/kg (preferably 0.01 mg/kg) as the lower limit and 10 mg/kg (preferably 2 mg/kg) as the upper limit, and is administered 1 to 6 times a day depending on the symptoms. .

The biological function testing and experimental tumor treatment of the compounds of the present invention are specifically introduced in the following examples, but it should be understood that the protection scope of the present invention is not limited to these examples.

Example

Materials or reagents used herein are commercially available or prepared by synthetic methods generally known in the art.

Example 1: Cell Testing

Test the compound of the present invention (TMZ) on human glioma U87 and U251 cultured in vitro, human lymphocytoma Mino, human Burkitt's tumor Raji, human B lymphocyte tumor Ramos, human multiple myeloma peripheral blood B lymphocyte tumor RPMI8226, human diffuse tissue lymphoma SU-DHL-2, (ABC type) diffuse lymphoma TMD-8, human diffuse large cell lymphoma cell SU-DHL-6 and other human tumor cells inhibit the proliferation.

tumor cell lines

A total of 9 tumor cell lines were used in this experiment, as shown in Table 1 below:

Table 1. List of cell lines

Cells were routinely cultured according to the method in Table 2.

Table 2. Cell culture conditions

On day 0 (D0) of the plating experiment, cells in the logarithmic growth phase were collected, prepared into a single cell suspension and counted, the cell concentration was adjusted to the required concentration, and 100 μL per well was added to a 96-well cell culture plate. The number of cells plated and cell concentration are shown in Table 3.

Table 3. Cell plating information

Drug Preparation:

U87, U251: Dissolve TMZ API (hereinafter referred to as TMZ) with DMSO and dilute it to 364.5μM, 121.5μM, 40.5μM, 13.5μM, 4.5μM, 1.5μM, 0.5μM, 0.167μM, 0.056μM as drug stock solution, and store in the refrigerator Refrigerate at 4°C until ready to use.

SU-DHL-2, Mino, Raji, Ramos, RPMI 8226, TMD-8, SU-DHL-6: Add an appropriate volume of DMSO and vortex to completely dissolve TMZ to prepare a stock solution with a concentration of 243mM, and dilute it to 486μM and 162μM. , 54.0μM, 18.0μM, 6.00μM, 2.00μM, 0.667μM, 0.222μM, 0μM, stored in -90℃～-60℃ refrigerator.

Detection and data analysis

IC50 detection

On D7, CCK8 solution was used for U87 and U251, and the Alamarblue method was used for the other 7 strains to detect the fluorescence intensity of each well, and the IC50 was calculated.

data analysis

IC50 is calculated by the following formula

Y＝Min+(Max-Min)/[1+〖10〗^((LogIC_50-X)*Slope)]

Min, Max and Slope represent the minimum value, maximum value and slope respectively.

QC

Any experimental phenomena, problems, and data generated during the experiment should be recorded in detail. The entire process of this experiment, all raw data, records, and bills generated.

The test results are shown in Table 4.

Table 4. The inhibitory effect of the compounds of the present invention on the proliferation of 9 human tumor cell lines

The data in Table 4 show that the compounds of the present invention only have good anti-tumor effects on brain gliomas and diffuse lymphocytomas, but have poor activity on other lymphomas or leukemia cells.

Example 2: Inhibitory effect of TMZ on tumor growth in vivo of human diffuse tissue lymphoma SU-DHL-2 xenograft tumor model

Purpose of the test: To test the inhibitory effect of TMZ compared to doxorubicin on tumor growth in the human diffuse tissue lymphoma SU-DHL-2 nude mouse transplanted tumor model in vivo.

Reagents: RPMI-1640 powder (Gibco; Cat. No.: 31800-022), fetal bovine serum (FBS, Gibco; Cat. No.: 10099-141C), cyan-streptobis antibody (HyClone; Cat. No.: SV30010; Lot No.: J220018), PBS ( 0.01M, pH 7.4, Sangon Biotech; Cat. No.: B040100-0005), N-N-dimethylacetamide (aladdin; Cat. No.: D108095), sterile water for injection (Zhejiang Dubang Pharmaceutical).

experimental animals

Female NOD/SCID mice (number: 62; age: 6-7 weeks) were purchased from Viton Lever and raised in the SPF animal room of Suzhou Shengsu New Drug Development Co., Ltd. at a temperature of 20-25°C and a relative humidity of 40 % ~ 70%, with light and dark lighting for 12 hours each; animals can drink water and eat freely. After about 5 days of normal feeding, mice with good physical condition after veterinary examination can be selected for this experiment. Before grouping, use a marker to mark the base of the animal's tail. After grouping, each animal will be marked with an ear cutout.

transplanted tumor lines

Human diffuse tissue lymphoma cells SU-DHL-2 were obtained from Kebai Biotech (liquid nitrogen cryopreservation in our laboratory).

cell culture

Under 5% CO ₂ and 37°C culture conditions, SU-DHL-2 cells were routinely cultured in RPMI-1640 culture medium containing 10% fetal bovine serum; they were passaged according to the cell growth conditions, with a passage ratio of 1:2 to 1:5.

Animal model preparation

Collect SU-DHL-2 cells in the logarithmic growth phase, count the cells and resuspend them in serum-free RPMI-1640 culture medium. Adjust the cell concentration to 1.5×10 ⁸ cells/mL; pipette the cells with a pipette to disperse them evenly. Put it into a 50-mL centrifuge tube, and place the centrifuge tube in an ice box; use a 1-mL syringe to draw the cell suspension, and inject it into the subcutaneous skin of the axilla of the front right limb of the mouse. Each animal is inoculated with 100 μL (1.5 × 10 ⁷ cells/ only) to establish a SU-DHL-2 mouse xenograft tumor model. After inoculation, the animal status and tumor growth were regularly observed, the tumor diameter was measured using an electronic vernier caliper, and the data was input into an Excel spreadsheet to calculate the tumor volume. When the tumor volume reached 100-300mm ³ , 42 animals in good health and with similar tumor volumes were selected and divided into 6 groups (n=7) using the random block method. The day of grouping was regarded as the first day of the experiment (D1). After the start of the experiment, the tumor diameter was measured twice a week, the tumor volume was calculated, and the animal body weight was weighed and recorded.

The calculation formula of tumor volume (TV) is as follows:
TV (mm ³ )＝l×w ² /2

Among them, l represents the long diameter of the tumor (mm); w represents the short diameter of the tumor (mm).

Preparation of dosing preparations

Blank solvent preparation

Take appropriate volumes of DMA and sterile water for injection respectively, mix them evenly, use them as blank solvents, and store them at room temperature for later use. The proportions of DMA and sterile water for injection in the mixed solvent are 10% and 90%.

Preparation of TMZ dosing preparations

Before each administration, weigh an appropriate amount of TMZ, add an appropriate volume of DMA to it, vortex to completely dissolve the compound, add an appropriate amount of sterile water for injection, vortex to mix the liquid evenly, and then add it to the dosage preparation. The proportions of DMA and sterile water for injection were 10% and 90%, and the final concentrations of the dosage preparations were 1.0, 2.0 and 3.0 mg·mL ^-1 respectively.

Animal grouping and drug administration

The animal grouping and dosing schedule are shown in Table 5. The test samples were administered starting on the day of grouping (D1), and the dosing period lasted for 3 weeks (the specific dosing time can be adjusted according to the progress of the experiment). The dosage, route and time of administration of the above compounds can be adjusted in a timely manner according to the progress of the experiment.

Table 5. Dosage regimen for drug efficacy experiments in mouse transplanted tumor model

Note: “ig” means intragastric administration, “QD” means once a day, and “QW” means once a week.

Experiment ends

On the last day of the experiment, the body weight and tumor diameter were measured, and the animals were euthanized by CO ₂ inhalation. The tumor pieces were removed, weighed, and photographed.

Data records, calculation formulas

The relative tumor volume (RTV) is calculated as:
RTV＝100×TV _t /TV _initial

Among them, TV _initial is the tumor volume measured during group administration; TV _t is the tumor volume measured at each time during the administration period.

The calculation formula of relative tumor proliferation rate (%T/C) is:
%T/C=100%×(RTV _T /RTV _C )

Among them, RTV _T represents the RTV of the treatment group; RTV _C represents the RTV of the solvent control group.

The calculation formula of tumor growth inhibition rate TGI (%) is:
TGI(%)＝100%×[1–(TV _t(T) –TV _initial(T) )/(TV _t(C) –TV _initial(C) )]

Among them, TV _{t (T)} represents the tumor volume measured each time in the treatment group; TV _{initial (T)} represents the tumor volume in the treatment group when administered in groups; TV _{t (C)} represents the tumor volume measured each time in the solvent control group; TV _initial(C) represents the tumor volume of the solvent control group during group administration.

The calculation formula of animal relative body weight (RBW) is:
RBW＝100×BW _t /BW _initial

Among them, BW _initial is the body weight of the animals during group administration; BW _t is the body weight of the animals measured each time during the administration period.

The calculation formula for animal weight loss rate is:

Animal weight loss rate = 100% × (BW _initial - BW _t )/BW _initial

Among them, BW _t represents the animal body weight measured each time during the administration period; BW _initial represents the animal body weight during group administration.

The calculation formula of tumor weight and tumor inhibition rate IR (%) is:
IR (%) = 100% × (W _C - W _T )/W _C

Among them, _WC represents the tumor weight in the solvent control group; _WT represents the tumor weight in the treatment group.

Statistical analysis methods

Experimental data were calculated and related statistical processing using Microsoft Office Excel 2007 software. Unless otherwise stated, data are expressed as mean±standard error (Mean±SE), and t-test was used for comparison between two groups.

Experimental observation

During the experiment, experimenters and veterinarians need to continuously observe the physical signs and health status of the experimental animals. Any abnormal performance of the animal, such as pain, depression, reduced activity, etc., must be recorded in the original experimental record. If the abnormal performance of experimental animals exceeds the provisions of IACUC-related animal welfare documents, the veterinarian can determine whether to terminate the experiment.

Experimental results

The effects on the growth of transplanted tumors in SU-DHL-2 nude mice are shown in Table 6.

Table 6 Average tumor volume ± SE (mm ³ ) and tumor growth inhibition rate (TGI) of animals in each group during the administration period

Note: “*” indicates that there is a significant difference between the tumor volume and the solvent control group (P<0.05), and “**” indicates that there is a highly significant difference between the tumor volume and the solvent control group (P<0.01).

Group A was the solvent control group. The animals were given blank solvent by gavage. The tumors of the animals grew well and no spontaneous regression was found. By D22, the average tumor volume reached 1163±133mm ³ .

Group B is the positive control drug doxorubicin group. Severe toxicity occurred at the dose of 7.5 mg·kg ^-1 QW. The animals' weight initially dropped severely and then all died. The tumor growth inhibition rate on D11 was 109% (P< 0.01), which has a significant inhibitory effect on tumor growth.

Group C is the TMZ low-dose group, with a dose of 10 mg·kg ^-1 QD. The tumor growth inhibition rate on D22 was 114% (P<0.05), showing an extremely excellent anti-tumor effect. By D11, the anti-tumor effect was already better than The safety of the positive control drug is significantly better than that of the positive control drug group.

Group D is the TMZ medium-dose group, with a dose of 20 mg·kg ^-1 QD. Although the tumor growth inhibition rate on D22 is the same as that of the low-dose group, both 114% (P<0.05), it has a faster onset of action than the low-dose group. It showed an extremely excellent anti-tumor effect. By D8, the anti-tumor effect had reached that of the positive control drug. By D11, the anti-tumor effect had been better than that of the positive control drug, and its safety was significantly better than that of the positive control drug group.

Group E is a high-dose TMZ group, with a dose of 30 mg·kg ^-1 QD. Although the tumor growth inhibition rate was 116% on D22 (P<0.05), the tumors completely disappeared, and the effect was faster than that of the low- and medium-dose groups. It showed extremely excellent anti-tumor effect, especially by D4, the anti-tumor effect was better than that of the positive control drug, and its safety was significantly better than that of the positive control drug group.

The effects on the body weight of SU-DHL-2 tumor-bearing mice are shown in Table 7.

Table 7 Body weight and weight loss rate of animals in each group during the administration period

Note: “-” means the animal’s weight has increased.

During the administration period, the animals in the solvent control group (Group A) and the low, medium and high TMZ groups (Groups C, D, and E) were in good condition. By D22, their body weight increased by 7.5%, 2.1%, -4.4% and -11% respectively. The positive control drug doxorubicin group (group B) had significant weight loss in the early stage. On D15, all animals died due to excessive drug dosage.

It can be seen from Tables 6-7 above that the compounds of the present invention are not only more effective but also safer than the commonly used anti-lymphoma drug doxorubicin at extremely high doses.

Example 3: Inhibitory effect of TMZ on tumor growth in vivo of human diffuse tissue lymphoma TMD-8 xenograft tumor model

Purpose of the test: In this experiment, cyclophosphamide was used as a control and the human diffuse large B lymphoma TMD-8 mouse transplant tumor model was used to evaluate the anti-tumor effect of TMZ.

Reagents: RPMI-1640 powder (Gibco; Cat. No.: 31800-022); Fetal bovine serum FBS (Gibco; Cat. No.: 10099-141C); HyClone; Cat. No.: SV30010); PBS (Beyotime; Cat. No.: ST447 ); Sterile water for injection (Zhejiang Dubang Pharmaceutical); Normal saline (Guizhou Tiandi Pharmaceutical); N-N-dimethylacetamide (aladdin; Cat. No.: D108095).

experimental animals

Female CB17/SCID mice (number: 75; age: 6-8 weeks) were purchased from Viton Lever and raised in the SPF animal room of Suzhou Shengsu New Drug Development Co., Ltd. at a temperature of 20-25°C and a relative humidity of 40 % ~ 70%, with light and dark lighting for 12 hours each; animals can drink water and eat freely. After 7 days of normal feeding, mice with good physical condition after veterinary examination can be selected for this experiment. Before grouping, use a marker to mark the base of the animal's tail. After grouping, each animal will be marked with an ear cutout.

transplanted tumor lines

Human diffuse large B lymphoma TMD-8 cells were purchased from Shanghai Huzhen Industrial Co., Ltd. and were frozen in liquid nitrogen in our laboratory.

cell culture

Under the conditions of 5% CO ₂ , 37°C, and saturated humidity, TMD-8 cells were routinely cultured in RPMI-1640 culture medium containing 10% fetal bovine serum. According to the cell growth conditions, they were digested and passaged with 0.25% trypsin. Passage 1 to 2 times per week, with a passage ratio of 1:3 to 1:4.

Animal model preparation

Collect TMD-8 cells in the logarithmic growth phase, count the cells, and resuspend them in a culture medium containing 30% serum-free RPMI-1640 and 70% Matrigel. Adjust the cell concentration to 5.0×10 ⁷ cells/mL; place the cells on ice. In the box, use a 1-mL syringe to draw the cell suspension and inject it into the subcutaneous tissue of the axilla of the front right limb of the mouse. Each animal is inoculated with 200 μL (1.0 × 10 ⁷ cells/mouse) to establish a TMD-8 transplanted tumor model. Observe the status of the animals regularly, use electronic vernier calipers to measure the tumor diameter, input the data into an Excel spreadsheet, calculate the tumor volume, and monitor tumor growth. When the tumor volume reaches 100-300mm ³ , 48 tumor-bearing mice in good health with similar tumor volumes are selected and divided into 6 groups (n=8) according to the tumor volume using random block method. At the same time, try to ensure that the average weight of each group is maintained. consistent. The day of grouping was regarded as the first day of the experiment (D1). After the start of the experiment, the tumor diameter was measured twice a week, the tumor volume was calculated, and the animal body weight was weighed and recorded.

Preparation of dosing preparations

Blank solvent preparation

Take appropriate volumes of DMA (N-N-dimethylacetamide) and sterile water for injection respectively, mix them evenly, use them as blank solvents, and store them at room temperature for later use. The proportions of DMA and sterile water for injection in the mixed solvent are 10% and 90% respectively.

Preparation of TMZ dosing preparations

Before each administration, weigh an appropriate amount of TMZ, add an appropriate volume of DMA to it, vortex, and sonicate for 10-20 minutes to disperse the compound evenly, add an appropriate amount of sterile water for injection, and vortex to completely dissolve the drug. The proportions of DMA and sterile water for injection in the dosage preparation are 10% and 90%, and the dosage preparations with final concentrations of 0.125, 0.25 and 0.5 mg/mL are obtained respectively, which are prepared for immediate use.

Preparation of CTX dosing preparations

Weigh an appropriate amount of CTX and put it into a glass bottle; add an appropriate amount of normal saline to the bottle, vortex and ultrasonic for 15 minutes to completely dissolve the drug; prepare a dosage preparation with a concentration of 2.5 mg/mL before each administration. Preparation, ready for use.

Animal grouping and drug administration

The animal grouping and dosing schedule are shown in Table 8. The first dose was started on the day of grouping (D1), and the dosing volume was 10 mL/kg.

Table 8. Dosage regimen for drug efficacy experiments in mouse transplanted tumor model

Note: “iv” means intravenous injection, “ig” means intragastric administration, “QD” means once a day, and “QW” means once a week.

The end-of-experiment processing, data recording, calculation formulas, statistical analysis methods, and experimental observations are the same as those in Example 2.

Experimental results

The effects on the growth of transplanted tumors in TMD-8 nude mice are shown in Table 9.

Table 9 Average tumor volume ± SE (mm ³ ) and tumor growth inhibition rate (TGI) of animals in each group during the administration period

Note: “*” indicates that there is a significant difference between the tumor volume and the solvent control group (P<0.05), and “**” indicates that there is a highly significant difference between the tumor volume and the solvent control group (P<0.01).

The effects on the body weight of TMD-8 tumor-bearing mice are shown in Table 10.

Table 10 Body weight and weight loss rate of animals in each group during the administration period

Note: “-” means the animal’s weight has increased.

The experimental results in Table 9 show that TMZ at doses of 1.25, 2.5 and 5.0 mg/kg (ig, QD), the tumor volume inhibition rate TGI was greater than 60%, and p<0.01, showing significant inhibition on the growth of transplanted tumors. The effective dose is lower than 1.25 mg/kg (ig, QD); TMZ has a significant inhibitory effect on the growth of human diffuse large B lymphoma TMD-8 transplanted tumor at a dose of 1.25 mg·kg ^-1 (ig, QD). Better than CTX (25mg·kg-1,iv,QW) dose. The anti-tumor efficacy of the combination of TMZ (1.25mg/kg, ig, QD) + CTX (25mg/kg, iv, QW) is significantly better than that of CTX (25mg/kg, iv, QW) alone.

The experimental results in Table 10 show that TMZ had no significant effect on the body weight of human diffuse large B lymphoma TMD-8 tumor-bearing mice at the dose set in this experiment, and no obvious drug-related abnormal reactions were observed during the administration; TMZ (1.25mg/kg, i.g., QD) combined with CTX (25mg/kg, i.v., QW) has a weight loss rate of less than 5%, which also shows good safety.

It can be seen from the above Tables 9-10 that compared with the positive control drug CTX, the compound of the present invention is not only effective but also has better safety.

Example 4: Inhibitory effect of TMZ and its preparations on tumor growth in vivo of human diffuse tissue lymphoma SU-DHL-6 xenograft tumor model

Purpose of the test: This experiment used cyclophosphamide as a control and used the human diffuse large B lymphoma SU-DHL-6 mouse transplant tumor model to evaluate the anti-tumor effects of TMZ and its preparations alone and in combination with cyclophosphamide.

Reagents: RPMI-1640 powder (Gibco; Cat. No.: 31800-022); Fetal bovine serum FBS (Gibco; Cat. No.: 10099-141C); HyClone; Cat. No.: SV30010; Lot No.: J220018); PBS (Sangon) Biotech; Cat. No.: B040100-0005); Sterile water for injection (Zhejiang Dubang Pharmaceutical); Normal saline (Guizhou Tiandi Pharmaceutical); N-N-dimethylacetamide (aladdin).

experimental animals

Female NOD/SCID mice (number: 85; age: 6-8 weeks) were purchased from Viton Lever and kept in the SPF animal room of Suzhou Shengsu New Drug Development Co., Ltd. at a temperature of 20-25°C and a relative humidity of 40 % ~ 70%, with light and dark lighting for 12 hours each; animals can drink water and eat freely. After 7 days of normal feeding, mice with good physical condition after veterinary examination can be selected for this experiment. Before grouping, use a marker to mark the base of the animal's tail. After grouping, each animal will be marked with an ear cutout.

transplanted tumor lines

Human large cell lymphoma cells SU-DHL-6 were obtained from the Kunming Cell Bank of the Chinese Academy of Sciences (CAS(KM), cryopreserved in liquid nitrogen in our laboratory).

cell culture

Under the conditions of 5% CO ₂ , 37°C, and saturated humidity, SU-DHL-6 cells were routinely cultured in RPMI-1640 culture medium containing 10% fetal bovine serum; according to the cell growth conditions, they were digested and passaged with 0.25% trypsin. , passaged 1 to 2 times a week, with a passage ratio of 1:3 to 1:4.

Animal model preparation

Collect SU-DHL-6 cells in the logarithmic growth phase, count the cells and resuspend them in culture medium containing 50% serum-free RPMI-1640 and 50% Matrigel. Adjust the cell concentration to 3.0×10 ⁷ cells/mL; place the cells In an ice box, use a 1-mL syringe to draw the cell suspension, and inject it into the mouse's front right armpit subcutaneously. Each animal is inoculated with 200 μL (6.0 × 10 ⁶ cells/mouse) to establish a SU-DHL-6 transplanted tumor model. Observe the status of the animals regularly, use electronic vernier calipers to measure the tumor diameter, input the data into an Excel spreadsheet, calculate the tumor volume, and monitor tumor growth. When the tumor volume reaches 100-300mm ³ , 48 tumor-bearing mice in good health and with similar tumor volumes are selected and divided into 7 groups (n=8) according to the tumor volume using random block method. At the same time, try to ensure that the average weight of each group is maintained. consistent. The day of grouping was regarded as the first day of the experiment (D1). After the start of the experiment, the tumor diameter was measured twice a week, the tumor volume was calculated, and the animal body weight was weighed and recorded.

Preparation of dosing preparations

Preparation of dosing formulations of TMZ formulations

Before each administration, take out 1 TMZ capsule (i.e., the aforementioned TMZ preparation, the same below), take out the contents into a glass bottle, add 75 ml of sterile water for injection, vortex, and ultrasonic for 20 to 30 minutes to prepare a mixture containing 0.5 mg/ml (API) dosage preparation; then add an appropriate volume of sterile water for injection, and dilute to obtain dosage preparations with final concentrations of 0.125, 0.25 and 0.5 mg/mL respectively, which are ready for use.

The preparation of TMZ raw material drug delivery preparation, cyclophosphamide (CTX) delivery preparation and blank vehicle are the same as in Example 3.

Animal grouping and drug administration

The animal grouping and dosing schedule are shown in Table 11. The first dose was started on the day of grouping (D1), and the dosing volume was 10 mL/kg.

Table 11. Dosage regimen for drug efficacy experiments in mouse transplanted tumor model

Note: “i.v.” means intravenous injection, “i.g.” means intragastric administration, “QD” means once a day, and “QW” means once a week.

Experimental results

The effects on the growth of transplanted tumors in SU-DHL-6 nude mice are shown in Table 12.

Table 12 Average tumor volume ± SE (mm ³ ) and tumor growth inhibition rate (TGI) of animals in each group during the administration period

Note: “*” indicates that there is a significant difference between the tumor volume and the solvent control group (P<0.05), and “**” indicates that there is a highly significant difference between the tumor volume and the solvent control group (P<0.01).

The effects on the body weight of SU-DHL-6 tumor-bearing mice are shown in Table 13.

Table 13 Body weight and weight loss rate of animals in each group during the administration period

Note: “-” means the animal’s weight has increased.

The experimental results in Table 12 and Table 13 show that the use of TMZ raw material and TMZ preparation alone, as well as the combination of TMZ preparation and cyclophosphamide, at the doses set in this experiment, have an impact on the body weight of human large cell lymphoma SU-DHL-6 tumor-bearing mice. There was no obvious effect, and no obvious drug-related abnormal reactions were observed during administration. Cyclophosphamide (CTX) at a dose of 25 mg/kg (iv, QW) has no obvious inhibitory effect on the growth of transplanted tumors, and the tumor volume inhibition rate TGI is less than 60%, while TMZ preparations at 1.25, 2.5 and 5.0 mg/kg ( ig, QD) dose, has a significant inhibitory effect on the growth of transplanted tumors, and the tumor volume inhibition rate TGI is greater than 60%, and p<0.01; TMZ preparation (1.25mg/kg, ig, QD) and CTX (25mg/ kg, iv, QW) combination therapy has a significant inhibitory effect on the growth of transplanted tumors, and the tumor inhibition rate in the combination group is significantly higher than that of TMZ preparation (1.25 mg/kg, ig, QD) and CTX (25 mg/kg, iv, QW) Used alone, it shows obvious synergistic and/or additive effects; TMZ has a significant inhibitory effect on the growth of transplanted tumors at a dose of 5 mg/kg (ig, QD). Although the final tumor inhibitory effect is equivalent to that of TMZ (2.5 mg/kg, ig, QD), it has a faster onset of action. TMZ raw material drug has a significant inhibitory effect on the growth of transplanted tumors at a dose of 2.5 mg/kg (ig, QD), which is equivalent to the TMZ preparation (2.5 mg/kg, ig, QD) alone group.

Example 5: Inhibitory effect of TMZ on tumor growth in human brain glioma cell U87 transplanted tumor model in vivo

Test purpose: To test the inhibitory effect of TMZ on the in vivo tumor growth of human glioma cell U87 nude mouse transplanted tumor model.

Test instruments: centrifuge, American Thermo Company, model ThermoSorvall ST 40; Hitachi 7600 fully automatic biochemical analyzer equipped with corresponding reagents; electronic balance Sartorius Company, model: BSA3202S-CW; electronic balance, METTLER-TOLEDO, model: LE204; small animals Weight balance, Shanghai Yueping Scientific Instrument Co., Ltd., model: YP1002; pipette: eppendorf; IVC mouse cage, product of Suzhou Teaching Cage Factory.

Test site and U87 cells: Beijing Yongxin Kangtai Technology Development Co., Ltd.

Preparation of tumor-bearing mouse model:

U87 cells are human malignant glioma cells. The normal cell morphology is epithelial and grows adherently. Medium DMEM 90%, Fetal Bovine Serum 10%; culture conditions 37℃ 5% CO2; digestion conditions 0.25% (w/v) Trypsin-0.53mMEDTA solution, digestion time 5-10min; passage ratio 1:2 ~ 1:4 ; Change the fluid every 2 to 3 days. until the vaccination quantity is reached. The tumors were inoculated into nude mice and passaged twice. After the second passage, the tumors were removed and used for formal experiments. Reagents: RPMI-1640 culture medium, fetal bovine serum (FBS), cyan-streptomycin, PBS (0.01M, pH 7.4), Matrigel, sterile water for injection.

Take nude mice with a body weight of 16±2g and adaptively raise them in the experimental animal center for one week. Select tumor-bearing mice with vigorous tumor growth and no ulceration, and sacrifice them by cervical dislocation. The animals are disinfected with alcohol under sterile conditions on a clean bench. Skin, cut the skin, peel off the tumor. Cut the tumor tissue into about ^1.5mm3 and inoculate it subcutaneously in the right armpit of the animal with a trocar.

After the tumor is inoculated into nude mice, use a vernier caliper to measure the diameter of the transplanted tumor (long diameter a, short diameter b) and calculate the tumor volume. The calculation formula of tumor volume (TV) is: V=1/2×a×b ² , when the tumor volume grows to about 100mm3, the animals are randomly divided into 2 groups, each group has 8 animals, which are the control group and the TMZ medication group. The animals in each group are weighed twice a week and measured twice a week after starting the medication. Tumor volume.

Test drug: It is a white powder. It is provided by Beijing Zimeng Pharmaceutical Co., Ltd. and the dosage is calculated. The dosage is 40 mg/kg, which is equivalent to the dosage of clinical patients. Store it dry and protected from light.

Dosing method: Oral administration, once a day; Dosing cycle: 19 days; Solvent: Animal drinking pure water

Calculation and statistical methods: After the experiment, the tumor tissue was taken, weighed, and the weight of the isolated tumor was measured. The average body weight, average tumor volume, and tumor inhibition rate of each group were calculated.

The relative tumor volume (RTV) is calculated based on the measurement results. The calculation formula is: RTV=Vt/V0, where V0 is the tumor volume measured when the cage is administered (i.e. d0), and Vt is the tumor volume at each measurement. Tumor volume.

Tumor growth inhibition rate (%), the calculation formula is as follows:

Animal grouping and drug administration

The animal grouping and dosing schedule are shown in Table 14. The first dose will be started on the day of grouping (D1).

Table 14. Dosage regimen for drug efficacy experiments in mouse transplanted tumor model

Experimental results

The effects on the growth of transplanted tumors in U87 nude mice are shown in Table 15.

Table 15 Average tumor volume ± SE (mm ³ ) and tumor growth inhibition rate (TGI) of animals in each group during the administration period

Note: “*” indicates that there is a significant difference between the tumor volume and the solvent control group (P<0.05), and “**” indicates that there is a highly significant difference between the tumor volume and the solvent control group (P<0.01).

The effects on the body weight of U87 tumor-bearing mice are shown in Table 16.

Table 16 Body weight and weight loss rate of animals in each group during the administration period

Note: “-” means the animal’s weight has increased.

It can be seen from Tables 15-16 above that the compounds of the present invention have good anti-proliferative effects on glioma. During the administration period, the body weight of the animals in the TMZ group continued to be low, once reduced by 7.9%, and compared with the blank control group, the weight of the animals once decreased by approximately 15%.

Comprehensive Examples 1 to 5, although the in vitro test shows that the inhibitory effect of TMZ on glioma cells and large B lymphoma cells is equivalent and good, the in vivo effectiveness of TMZ on the two is greatly different: the dose is as low as 2.5 mg·kg ^-1 has produced significant anti-tumor activity against various types of DLBCL. At the end of the trial, the tumor volume was smaller than the initial volume, and even the tumor volume was close to disappearing. When the dose reached 30 mg·kg ^-1 , all tumors disappeared, and all animals The growth status was normal and there was no significant change in body weight; correspondingly, when the TMZ dose reached 40 mg·kg ^-1 , the glioma tumor volume still grew slowly, and some animals died, with a significant decrease in body weight. Considering that the glioma animal model is not intracranial inoculation, although TMZ has a significant advantage in its ability to penetrate the blood-brain barrier among anti-tumor drugs, its exposure in brain tissue is less than half of that in the circulation system. First, it can be reasonably presumed that TMZ is less effective on intracranial gliomas. Glioma although TMZ is the only globally approved Indications, but the effect and safety of TMZ on DLBCL far exceed that of glioma. For example, primary central nervous system lymphoma, also known as microglioma cells, only use TMZ's better blood-brain barrier penetration. ability, the use of TMZ has not produced significant effects (M Kazuhiko, N Ryo et al. Randomized phase III study of high-dose methotrexate and whole-brain radiotherapy with/without temozolomide for newly diagnosed primary CNS lymphoma: JCOG1114C, Neuro-Oncology, 1– 12, 2022). Therefore, the present invention mainly provides the use of TMZ in the treatment and/or prevention of DLBCL other than diffuse large B-cell lymphoma (DLBCL) whose primary site or metastatic site is the central nervous system.

Industrial applicability

The imidazotetrazine compounds proposed by the present invention have excellent antagonistic effects and excellent safety on diffuse lymphoma whose primary site or metastasis site is not the central nervous system.

Claims

Imidazotetrazine compounds or their solvates, hydrates, polymorphs, prodrugs or pharmaceutically acceptable salts thereof in the preparation of medicaments for the treatment and/or prevention of diffuse large B-cell lymphoma (DLBCL) Purpose, the imidazotetrazine compound is shown in formula I:

Wherein, R 1 is hydrogen or C1-C3 alkyl, R 2 is amino or amino substituted by C1-C3 alkyl; R 3 is hydrogen or C1-C3 alkyl.
Use of a pharmaceutical composition in the preparation of a medicament for the treatment and/or prevention of diffuse large B-cell lymphoma (DLBCL), the pharmaceutical composition comprising an imidazotetrazine compound represented by Formula I or a solvate thereof , hydrates, polymorphs, prodrugs or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable excipients,

Wherein, R 1 is hydrogen or C1-C3 alkyl, R 2 is amino or amino substituted by C1-C3 alkyl; R 3 is hydrogen or C1-C3 alkyl.
The use according to claim 1 or 2, wherein R 1 is methyl, R 2 is amino, and R 3 is hydrogen.
The use according to any one of claims 1 to 3, wherein the DLBCL is selected from diffuse large B-cell lymphoma whose primary site and/or metastatic site is not the central nervous system,

Preferably, the diffuse large B-cell lymphoma is selected from the group consisting of unspecified DLBCL, other large B-cell lymphomas, high-grade B-cell lymphomas, and unclassifiable B-cell lymphomas.
The use according to any one of claims 1 to 4, wherein the DLBCL is selected from the group consisting of centroblast variant DLBCL, immunoblastic variant DLBCL, anaplastic variant DLBCL, spindle cell variant DLBCL, and signet ring cell-like variant. DLBCL, T-cell/histiocyte-enriched DLBCL, primary cutaneous DLBCL, EBV-positive DLBCL, chronic inflammation-associated large B-cell lymphoma, lymphomatoid granuloma, large B-cell lymphoma with IRF4 rearrangement, Large B-cell lymphoma with IRF4/MUM1 rearrangement, primary mediastinal large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, HHV8-positive DLBCL, Primary effusion lymphoma, high-grade B-cell lymphoma with MYC, BCL2, and/or BCL6 rearrangements, high-grade B-cell lymphoma not otherwise specified, unclassifiable between DLBCL and classical Hodgkin lymphoma B-cell lymphoma, B-cell lymphoma between primary mediastinal large B-cell lymphoma and nodular sclerosing classical Hodgkin lymphoma, B-cell lymphoma between DLBCL and Burkitt lymphoma tumor;

Optionally, the DLBCL cells are selected from the group consisting of human diffuse tissue lymphoma cell SU-DHL-2 and human diffuse lymphoma cell line TMD-8. and one or more of human diffuse large cell lymphoma cells SU-DHL-6.
The use according to any one of claims 1 to 5, wherein the DLBCL is primary DLBCL, or transformed DLBCL, or late and/or relapsed, and/or refractory DLBCL, or in DLBCL that has first received systemic therapy, or DLBCL that has failed chemotherapy and/or targeted drugs and/or immunotherapy.
A pharmaceutical combination comprising an imidazotetrazine compound and a second therapeutic agent capable of treating DLBCL, the imidazotetrazine compound being represented by Formula I:

Among them, R 1 is hydrogen or C1-C3 alkyl, R 2 is amino or C1-C3 alkyl substituted amino; R 3 is hydrogen or C1-C3 alkyl.
The pharmaceutical combination according to claim 7, wherein R 1 is methyl, R 2 is amino; R 3 is hydrogen.
The pharmaceutical combination according to claim 7 or 8, wherein the second therapeutic agent is one or more of chemotherapy drugs, small molecule targeted anti-tumor drugs, immunotherapy drugs, and antibody drugs; preferably, The second therapeutic agent is cyclophosphamide.
The use of the pharmaceutical combination according to any one of claims 7 to 9 in the preparation of medicaments for the treatment and/or prevention of DLBCL.
The use according to claim 10, wherein the DLBCL is selected from DLBCL whose primary site and/or metastasis site is not central nervous system DLBCL,

Preferably, the DLBCL is selected from unspecified DLBCL, other large B-cell lymphomas, high-grade B-cell lymphomas, and unclassifiable B-cell lymphomas.
The use according to claim 10, wherein the DLBCL is selected from the group consisting of centroblast variant DLBCL, immunoblastic variant DLBCL, anaplastic variant DLBCL, spindle cell variant DLBCL, signet ring cell-like variant DLBCL, T cell/ Histiocyte-enriched DLBCL, primary cutaneous DLBCL, EBV-positive DLBCL, chronic inflammation-associated large B-cell lymphoma, lymphomatoid granuloma, large B-cell lymphoma with IRF4 rearrangement, with IRF4/MUM1 Rearranged large B-cell lymphoma, primary mediastinal large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, HHV8-positive DLBCL, primary effusion lymphoma, high-grade B-cell lymphoma with MYC, BCL2 and/or BCL6 rearrangements, high-grade B-cell lymphoma not otherwise specified, unclassifiable B-cell lymphoma between DLBCL and classical Hodgkin lymphoma , B-cell lymphoma between primary mediastinal large B-cell lymphoma and nodular sclerosis classical Hodgkin lymphoma, B-cell lymphoma between DLBCL and Burkitt lymphoma;

Optionally, the DLBCL cells are selected from the group consisting of human diffuse tissue lymphoma cell SU-DHL-2, human diffuse lymphoma cell line TMD-8 and human diffuse large cell lymphoma cell SU-DHL-6. one or more.
The use according to any one of claims 10 to 12, wherein the DLBCL is primary DLBCL, or transformed DLBCL, or late and/or relapsed, and/or refractory DLBCL, or in DLBCL that has first received systemic therapy, or DLBCL that has failed chemotherapy and/or targeted drugs and/or immunotherapy.
A method of treating and/or preventing DLBCL in a subject, the method comprising administering to the subject an effective amount of an imidazotetrazine compound or a solvate, hydrate, polymorph, prodrug or Its pharmaceutically acceptable salts or pharmaceutical combinations including them, the imidazotetrazine compounds are represented by formula I:

Among them, R 1 is hydrogen or C1-C3 alkyl, R 2 is amino or C1-C3 alkyl substituted amino; R 3 is hydrogen or C1-C3 alkyl.
The method according to claim 14, wherein R 1 is methyl, R 2 is amino; R 3 is hydrogen.
The method of claim 14, wherein the pharmaceutical combination further includes a second therapeutic agent capable of treating DLBCL.
The method according to claim 16, wherein the second therapeutic agent is one or more of a chemotherapy drug, a small molecule targeted anti-tumor drug, an immunotherapy drug, and an antibody drug. Preferably, the second therapeutic agent is The second therapeutic agent is cyclophosphamide.
The method according to any one of claims 14 to 17, wherein the DLBCL is selected from DLBCL whose primary site and/or metastasis site is not central nervous system DLBCL,

Preferably, the DLBCL is selected from unspecified DLBCL, other large B-cell lymphomas, high-grade B-cell lymphomas, and unclassifiable B-cell lymphomas.
The method according to any one of claims 14 to 17, wherein the DLBCL is selected from the group consisting of centroblast variant DLBCL, immunoblastic variant DLBCL, anaplastic variant DLBCL, spindle cell variant DLBCL, and signet ring cell-like variant. DLBCL, T-cell/histiocyte-enriched DLBCL, primary cutaneous DLBCL, EBV-positive DLBCL, chronic inflammation-associated large B-cell lymphoma, lymphomatoid granuloma, large B-cell lymphoma with IRF4 rearrangement, Large B-cell lymphoma with IRF4/MUM1 rearrangement, primary mediastinal large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, HHV8-positive DLBCL, Primary effusion lymphoma, high-grade B-cell lymphoma with MYC, BCL2, and/or BCL6 rearrangements, high-grade B-cell lymphoma not otherwise specified, unclassifiable between DLBCL and classical Hodgkin lymphoma B-cell lymphoma, B-cell lymphoma between primary mediastinal large B-cell lymphoma and nodular sclerosing classical Hodgkin lymphoma, B-cell lymphoma between DLBCL and Burkitt lymphoma tumor;

Optionally, the DLBCL cells are selected from the group consisting of human diffuse tissue lymphoma cell SU-DHL-2, human diffuse lymphoma cell line TMD-8 and human diffuse large cell lymphoma cell SU-DHL-6. one or more.
The method according to any one of claims 14 to 17, wherein the DLBCL is primary DLBCL, or transformed DLBCL, or late and/or relapsed, and/or refractory DLBCL, or in DLBCL that has first received systemic therapy, or DLBCL that has failed chemotherapy and/or targeted drugs and/or immunotherapy.